Cross-linked biopolymers, related compositions and methods of use

ABSTRACT

The present invention provides stabilized oil-in-water emulsions with an extended range of chemical, thermal and/or mechanical stabilities, and method(s) for their preparation. Such preparations provide an environmentally-protective biopolymer component exhibiting improved adherence to the dispersed phase, reducing or eliminating dissociation therefrom under such conditions, for use in the context of a range of food, pharmaceutical, personal care, health care, cosmetic and other end-use applications.

This application claims priority benefit of application Ser. No.61/008,389 filed Dec. 20, 2007, the entirety of which is incorporatedherein by reference.

The United States government has certain rights to this inventionpursuant to Award Nos. 2002-35503-12296 and 2005-35503-16164 from theUnited States Department of Agriculture to the University ofMassachusetts.

BACKGROUND OF THE INVENTION

Protein-stabilized oil-in-water emulsions are widely utilized in thefood, cosmetics and pharmaceutical industries. These emulsions consistof protein-coated lipid droplets dispersed in an aqueous continuum.Conventionally, these emulsions are created by homogenizing an oil phasewith an aqueous phase containing surface-active proteins, such as tocasein, soy proteins, egg proteins or whey proteins. The proteinmolecules adsorb to the surface of the droplets produced duringhomogenization where they form a protective coating that prevents themfrom aggregating, e.g., flocculating and/or coalescing. In addition, theadsorbed proteins reduce the oil-water interfacial tension, therebyfacilitating the further disruption of lipid droplets duringhomogenization and leading to smaller droplet sizes. At present,proteins can only be used successfully as emulsifiers in a limited rangeof materials because of their high sensitivity to changes in solution pHand ionic strength. Protein-coated lipid droplets are primarilystabilized by electrostatic repulsion, consequently they tend toaggregate when the pH moves close to the isoelectric point of theproteins (due to reduction of the ζ-potential) or when the ionicstrength increases above a certain level (due to increased electrostaticscreening). In addition, protein-coated lipid droplets are oftensusceptible to aggregation when they are heated above the thermaldenaturation temperature of the adsorbed proteins because this increasesthe hydrophobic attraction between them.

Recently, an interfacial engineering technology, based on thelayer-by-layer (LbL) electrostatic deposition technique, has been usedto improve the stability of protein-coated lipid droplets toenvironmental stresses, such as pH, ionic strength and temperature.Initially, a “primary emulsion” consisting of lipid droplets coated by alayer of charged globular proteins is produced using conventionalhomogenization. Then, a “secondary emulsion” is formed by depositing anoppositely charged polyelectrolyte (e.g., an ionic polysaccharide) ontothe surface of the protein-coated lipid droplets. This procedure can berepeated a number of times by successively depositing layers ofoppositely charged polyelectrolytes onto the surfaces of the lipiddroplets so that multilayered interfacial coatings are formed. Rationalselection of polyelectrolyte characteristics and deposition conditionsenables one to carefully control interfacial characteristics, such asthickness, charge, permeability, environmental responsiveness andfunctionality.

One potential limitation of the electrostatic deposition method forcertain applications is that interfacial protein-polysaccharidecomplexes are only held together by electrostatic attraction.Consequently, the polysaccharide layer may dissociate from theprotein-coated lipid droplet surfaces when the pH is varied so that theprotein and polysaccharide have opposite charges, or when the ionicstrength is increased above a certain level. As a result, there remainsan on-going search in the art to provide emulsion systems that remainintact with changes in pH or ionic strength of the surrounding solution.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide stabilized oil-in-water emulsions and method(s) for theirpreparation, thereby overcoming various deficiencies of the prior artincluding those outlined above. It will be understood by those skilledin the art that one or more aspects of this invention can meet certainobjectives, while one or more other aspects can meet certain otherobjectives. Each objective may not apply equally, in all its respects,to every aspect of this invention. As such, the following objects can beviewed in the alternative with respect to any one aspect of thisinvention.

It can be an object of the present invention to enhance the physicalstability of such emulsions to environmental stresses.

It can be another object of the present invention to extend the range ofchemical, thermal and/or mechanical conditions over which such emulsionscan be utilized without either degradation or aggregation.

It can be another object of the present invention to provide anenvironmentally-protective biopolymer component exhibiting improvedadherence to the dispersed phase, reducing or eliminating dissociationtherefrom under such conditions.

It can also be an object of the present invention, alone or inconjunction with one or more of the preceding objectives, to providestabilized oil-in-water emulsions and/or methods for their preparation,for use in the context of a range of food, pharmaceutical, personalcare, health care, cosmetic and/or other end-use applications.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments, and will be readily apparent tothose skilled in the art having knowledge of such emulsions, relatedprocesses and applications. Such objects, features, benefits andadvantages will be apparent from the above as taken into conjunctionwith the accompanying examples, data, figures and all reasonableinferences to be drawn therefrom.

In part, the present invention can be directed to a compositioncomprising a hydrophobic fat/oil component; an emulsifier componentabout the hydrophobic component; and a biopolymer component adsorbed on,electrostatically, interactive with and/or coupled to the hydrophobiccomponent and/or the emulsifier component, such a biopolymer componentcomprising an intra-component covalent cross-linkage, such a linkage ascan be considered the reaction product of cross-linkable moieties and across-linking agent/reagent.

Without limitation, a hydrophobic component of such embodiments can beat least partially insoluble in an aqueous or another medium and/or iscapable of forming emulsions in an aqueous medium. In certain suchembodiments, a hydrophobic component can comprise a fat, an oil or acomponent thereof, including but not limited to any edible food oilknown to those skilled in the art (e.g., without limitation, corn,soybean, canola, rapeseed, olive, peanut, algal, palm, coconut, nutand/or vegetable oils, fish oils or combinations thereof). A hydrophobiccomponent can be selected from hydrogenated or partially hydrogenatedfats and/or oils, and can include any dairy or animal fat or oilincluding, for example, dairy fats. In addition, the hydrophobiccomponent can further comprise flavors, antioxidants, nutrients,nutraceuticals, preservatives and/or nutritional components, such as fatsoluble vitamins.

It will be readily apparent that, consistent with the broader aspects ofthe invention, a hydrophobic component can further include any naturaland/or synthetic lipid components including, but not limited to, fattyacids (saturated or unsaturated), glycerols, glycerides and theirrespective derivatives, phospholipids and their respective derivatives,glycolipids, phytosterol and/or sterol esters (e.g., cholesterol esters,phytosterol esters and derivatives thereof), carotenoids, terpenes,antioxidants, colorants, and/or flavor oils (for example, peppermint,citrus, coconut, or vanilla and extracts thereof such as terpenes fromcitrus oils), as may be required by a given food or beverage end useapplication. Other such components include, without limitation,brominated vegetable oils, ester gums, sucrose acetate isobutyrate,damar gum and the like. The present invention, therefore, contemplates awide range of edible oil/fat, waxes and/or lipid components of varyingmolecular weight and comprising a range of hydrocarbon (aromatic,saturated or unsaturated), alcohol, aldehyde, ketone, acid and/or aminemoieties or functional groups.

An emulsifier component can comprise any food-grade surface activeingredient, cationic surfactant, anionic surfactant and/or amphiphilicsurfactant known to those skilled in the art capable of at least partlyemulsifying the hydrophobic component in an aqueous medium and impartinga net charge to at least a portion thereof. Accordingly, such anemulsifier component can be selected from one or more small-moleculesurfactants, fatty acids, phospholipids, proteins and polysaccharides,and derivatives thereof. Such emulsifiers can further include one ormore of, but not limited to, lecithin, chitosan, modified starches,pectin, gums (e.g., locust bean gum, gum arabic, guar gum, etc.),alginic acids, alginates and derivatives thereof, and cellulose andderivatives thereof. Protein emulsifiers can include any one of thedairy proteins (e.g., without limitation, whey and casein), vegetableproteins (e.g., soy), meat proteins, fish proteins, plant proteins, eggproteins, ovalbumins, glycoproteins, mucoproteins, phosphoproteins,serum albumins, collagen and combinations thereof. Protein emulsifyingcomponents can be selected on the basis of their amino acid residues(e.g., lysine, arginine, asparatic acid, glutamic acid, etc.) tooptimize the overall net charge of the interfacial membrane about thehydrophobic component, and therefore the stability of the hydrophobiccomponent within an emulsion system.

Indeed, an emulsifier component can include a broad spectrum ofemulsifiers including, for example, acetic acid esters of monogylcerides(ACTEM), lactic acid esters of monogylcerides (LACTEM), citric acidesters of monogylcerides (CITREM), diacetyl acid esters ofmonogylcerides (DATEM), succinic acid esters of monogylcerides,polyglycerol polyricinoleate, sorbitan esters of fatty acids, propyleneglycol esters of fatty acids, sucrose esters of fatty acids, mono anddiglycerides, fruit acid esters, stearoyl lactylates, polysorbates,starches, sodium dodecyl sulfate (SDS) and/or combinations thereof.

Regardless, a biopolymer component can comprise any food-grade polymericmaterial capable of adsorption on, electrostatic interaction with and/orcoupling to a hydrophobic component and/or an associated emulsifiercomponent. Accordingly, such a biopolymer component can be selected fromone or more proteins, one or more polysaccharides and combinationsthereof. Without limitation, such a biopolymer component can be selectedfrom but not limited to food-grade ionic or ionizable proteins such aswhey, casein, soy, egg, plant, meat and fish proteins, ovalbumins,glycoproteins, mucoproteins, phosphoproteins, serum albumins andcollagens, and ionic or ionizable polysaccharides such as chitosanand/or chitosan sulfate, cellulose, pectins, alginic acids, alginates,glycogen, amylose, chitin, gum arabic, gum acacia, carageenans,xanthans, agars, tree gums and exudates thereof, guar gum, gellan gum,tragacanth gum, karaya gum, locust bean gum, lignin, nucleic acids,polynucleotides and/or combinations thereof. As mentioned above, suchprotein components can be selected on the basis of their amino acidresidues (e.g., lysine, arginine, aspartic acid, glutamic acid, etc.) tooptimize overall net charge, interaction with an emulsifier componentand/or resultant emulsion stability. The food-grade polymeric componentmay alternatively be selected from modified polymers such as modifiedstarch, modified celluloses, carboxymethyl cellulose, carboxymethyldextran or lignin sulfonates.

As mentioned above and described elsewhere herein, such abiopolymer—regardless of specific composition—can comprise covalentintra-component cross-linkages, as would be understood by those skilledin the art made aware of this invention. Such intra-component structurescan be achieved by introduction of and/or use of an agent or reagent atleast partially sufficient to at least partially cross-link thebiopolymer component(s). Such agents are as would be understood by thoseskilled in the art and include, without limitation, compounds, enzymes,heat, pH, and various redox reagents. In certain such embodiments,depending upon choice of biopolymer component and cross-linkable moietyor moieties present therewith, enzymatic cross-linking can be employed.Without limitation, various pectins can be, as described below,cross-linked by the activity of laccase and peroxidase enzymes.

In certain embodiments of this invention, an emulsifiercomponent—depending upon the presence of one or more cross-linkablemoieties—can also be covalently cross-linked under applicable reactionor process conditions employed. Without limitation, various wheyproteins can also be enzymatically cross-linked. Accordingly, in certainsuch compositional embodiments of this invention, the emulsifier andbiopolymer components can be covalently cross-linked, suchcross-linkages selected from covalent intra-component cross-linkages,covalent inter-component cross-linkages and combinations thereof.

The present invention contemplates any combination of emulsifier andbiopolymer components leading to the formation of a multi-layeredcomposition comprising an oil/fat and/or lipid hydrophobic componentsufficiently stable under environmental or end-use conditions (e.g., asapplicable to a particular food product). Accordingly, a hydrophobiccomponent can be at least partially coated or encapsulated with and/orimmobilized by a wide range of emulsifiers/polymeric components,depending upon the pH, ionic strength, salt concentration, temperatureand processing requirements of the system/food product into which ahydrophobic component is to be incorporated. Such anemulsifier/biopolymer component combinations are limited only byinteraction one with another and covalent intra- and/or inter-componentcross-linking thereof.

As illustrated herein, such compositions can be provided in the contextof an emulsion system: for example, as a dispersed phase in an aqueouscontinuous phase. The resulting emulsion compositions/products can bedehydrated (e.g., by spray- or freeze-drying), then reconstituted forlater use. Regardless, one or more such cross-linked compositions can beemployed with a range of end-use applications including but limited tofoods, beverages, pharmaceuticals, nutraceuticals, personal and healthcare products, agro-chemicals, cosmetics and the like.

In part, this invention can provide a biomimetic method for preparationand/or stabilizing an emulsified substantially hydrophobic oil/fatcomponent. Such a method can comprise providing a hydrophobic and/oroil/fat and/or lipid component; contacting the hydrophobic/oil/fatcomponent with one or more emulsifier components, at least a portion ofeach as can comprise a net charge; contacting or incorporating therewithone or more food-grade biopolymer components can comprise across-linkable moiety, at least a portion of each as can comprise a netcharge opposite that of the emulsifier component and/or a previouslyincorporated food-grade polymeric component; and introduction of anagent at least partially sufficient to at least partially covalentlycross-link the biopolymer component(s). Hydrophobic, emulsifier andbiopolymer components can be as known to those skilled in the art madeaware of this invention and/or selected as described above. In certainembodiments, such a method can comprise alternating contact orincorporation of oppositely charged emulsifier and/or food-gradebiopolymer components, each such contact or incorporation comprisingelectrostatic interaction with a previously contacted or incorporatedemulsifier or biopolymer component(s).

Regardless, cross-linking can be achieved by introduction of an agentincluding but not limited to an enzyme, compound, heat and/or pH and/orhydrogen ion concentration and various redox reagents, depending uponthe identity of any cross-linkable moiety present. In certainembodiments, without limitation, a biopolymer component can be apolysarcharide including but not limited to those selected from variousavailable pectins comprising ferulic acid moieties. In certain suchembodiments, such biopolymer components can cross-linked by introductionand activity of laccase and/or peroxidase enzymes thereon. In certainother embodiments, an emulsifier component—depending upon the presenceof one or more cross-linkable moieties—can also be covalentlycross-linked. Without limitation, such an emulsifier component can beselected from various available whey proteins comprising phenolic acidmoieties. Introduction and activity of laccase and/or peroxidase enzymesthereon can cross-link the corresponding whey protein. Accordingly, asdiscussed above, depending upon reaction or process conditions employed,the emulsifier and biopolymer components can be covalently cross-linked,such cross-linking selected from covalent intra-component cross-linking,covalent inter-component cross-linking and combinations thereof.

As illustrated herein, such preparation and/or cross-linking can be inan aqueous continuous phase. The resulting emulsion can be isolated,processed and/or incorporated into a range of food products, beverages,pharmaceuticals, nutraceuticals, personal and health care products,agro-chemicals, cosmetics and various other end-use applications, aswould be understood by those skilled in the art made aware of thisinvention.

In part, this invention can comprise an alternate method for emulsionand particulate formation. With reference to the preceding, a biopolymercomponent can be incorporated with or contact a composition comprisingan oil/fat component and an emulsifier component under conditions or ata pH not conducive for sufficient electrostatic interaction therewith.The pH can then be varied to change the net electrical charge of theemulsion, of the emulsified oil/fat/lipid component and/or of thepolymeric component, sufficient to promote electrostatic interactionwith and incorporation of the polymeric component. Without limitation, astable emulsion can be prepared using a protein emulsifier (e.g.,without limitation casein, whey, soy, egg or gelatin) at a pH below itsisoelectric point, to form cationic or net positively-charged emulsiondroplets, then using an anionic or net negatively-charged polysaccharide(e.g., without limitation, a pectin, carrageenan, alginate, or gumarabic) for electrostatic interaction with the initial emulsioncomposition. Regardless of method of preparation, such emulsions arestable to interaction with other emulsion components or with respect tofactors (e.g., pH, ionic strength, temperature, etc.) relating toend-use applications.

Without limitation, with reference to the following examples, emulsionscan be prepared using food-grade components and standard preparationprocedures (e.g., homogenization and mixing). Initially, a primaryaqueous emulsion comprising an electrically charged emulsifier componentcan be prepared by homogenizing an oil/fat component, an aqueous phaseand a suitable emulsifier comprising a net charge. Optionally,mechanical agitation or sonication can be applied to such a primaryemulsion to disrupt any floc formation, and emulsion washing can be usedto remove any non-incorporated emulsifier component. A secondaryemulsion can be prepared by contacting a net-charged biopolymercomponent with a primary emulsion. The polymeric component can have anet electrical charge opposite to at least a portion of the primaryemulsion. Optionally, mechanical agitation or sonication can also beapplied to disrupt any floc formation, and emulsion washing can be usedto remove any non-incorporated emulsifier component. As discussed above,emulsion characteristics can be altered by pH adjustment to promote orenhance electrostatic interaction of a primary emulsion and a biopolymercomponent.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. UV-visible absorption spectra of citrus and beet pectin (0.1 wt%) dissolved in aqueous phosphate buffer solution (5 mM buffer, pH 4.5).

FIGS. 2A-B. Influence of laccase concentration (0 to 10 AU) on (A) thetime-dependence of the UV-visible absorbance at 325 nm and (B) theinitial slope (1/A(0)×dA(t)/dt) for beet pectin (0.1 wt %) dissolved inaqueous phosphate buffer solution (5 mM buffer, pH 4.5).

FIG. 3. Dependence of particle charge (ξ-potential) on beet pectinconcentration for secondary emulsions: 1 wt % corn oil, 0.05 wt % β-Lg,0 to 0.1 wt % pectin, 5 mM phosphate, pH 4.5.

FIG. 4. Dependence of mean particle diameter (d₃₂) on beet pectinconcentration for secondary emulsions: 1 wt % corn oil, 0.05 wt % β-Lg,0 to 0.1 wt % pectin, 5 mM phosphate buffer, pH 4.5.

FIG. 5. Dependence of ζ-potential on pH and laccase concentration (0 or5 AU) for primary and secondary emulsions: 1 wt % corn oil, 0.05 wt %β-Lg, 0 or 0.04 wt % pectin, 5 mM phosphate buffer. The pH of theemulsions was sequentially adjusted from (1) pH 7 to (2) pH 4.5 to (3)pH 7.

FIG. 6. Dependence of mean particle diameter (z-average) on pH andlaccase concentration (0 or 5 AU) for primary and secondary emulsions: 1wt % corn oil, 0.05 wt % β-Lg, 0 or 0.04 wt % pectin, 5 mM phosphatebuffer. The pH of the emulsions was sequentially adjusted from (1) pH 7to (2) pH 4.5 to (3) pH 7.

FIGS. 7A-C. Influence of NaCl concentration on (A) the ζ-potential, (B)the mean particle diameter (d₃₂) and (C) the creaming stability ofprimary and secondary emulsions in the absence (0 AU) and presence (5AU) of laccase at pH 7. The primary emulsions were prepared and kept atpH 7.0, while the secondary emulsions were prepared at pH 7.0, adjustedto pH 4.5, then brought back to pH 7.0.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Illustrating other embodiments of this invention, an electrostaticdeposition method described previously was used to prepare emulsionscontaining lipid droplets coated by β-lactoglobulin-pectin layers.Representative of other embodiments, bovine β-Lactoglobulin (β-Lg) wasselected as an emulsifier to form the primary adsorbed layer around thelipid droplets because it is a surface-active globular protein with wellknown molecular and functional characteristics. Likewise, representativeof other embodiments, pectin was selected as a polysaccharide to formthe secondary adsorbed layer because it is an anionic biopolymer thathas previously been shown to adsorb to β-Lg-coated droplets at pH valuesbelow the isoelectric point of such proteins. Beet pectin was used inthis study because it can be covalently cross-linked by an enzyme(laccase). Beet pectin is extracted from sugar beet pulp, which is aby-product of the sugar industry. Unlike citrus pectins, beet pectinshave ferulic acid groups esterified to some of the neutral sugars in theside-chains of the so-called “hairy” regions. Oxidative enzymes, such aslaccase and peroxidase, can oxidise these ferulic acid groups through afree radical mechanism resulting in the formation of covalentcross-links between beet pectin molecules. Studies have shown thatlaccase can also cross-link whey proteins in the presence of phenolicacids. Therefore, laccase can be used to initiate protein-proteincross-links in the primary interfacial layer,polysaccharide-polysaccharide cross-links in the secondary interfaciallayer, and/or protein-polysaccharide cross-links between layers.

As demonstrated, below, stable emulsions containing lipid dropletscoated by β-lactoglobulin and beet pectin can be formed, with the beetpectin layer covalently cross-linked by laccase. Illustrating broaderapplication of this invention, such emulsions have improved stabilityover primary emulsions or secondary emulsions with no cross-linking.Mimicking biochemical processes prevalent in nature (such as enzymaticcross-linking of pectin) allows rational design of novel functionalperformance into commercial emulsified products (e.g., food products,etc.).

With reference to the following examples, laccase was used to crosslinkthe beet pectin molecules, and to ascertain the amount of such an enzymeneeded for adequate cross-linking within a certain timescale. Theability of laccase to crosslink the pectin molecules was establishedusing UV-visible absorption and gelation measurements.

At pH 4.5, there was no evidence of a peak in the absorption spectrum ofcitrus pectin from 200 to 800 nm, but there was an appreciable peakaround 320 to 330 nm in the absorption spectrum of the beet pectin (FIG.1). Previous studies have attributed this peak to absorption by ferulicacid groups. The difference in the absorption spectra of citrus and beetpectins can therefore be attributed to the greater amount of ferulicacid groups present in the latter. The height of the absorption peak dueto the ferulic acid groups changed when they were cross-linked bylaccase. Consequently, measurements of the absorption of pectinsolutions at 325 nm (A_(325nm)) were used to monitor the kinetics offerulic acid cross-linking by laccase at 25° C. The data is presented asthe normalized absorbance (A(t)/A(0)), i.e., the absorbance at time tdivided by the initial absorbance at time 0 (FIG. 2). The UV-visibleabsorption spectra of 0.5 wt % beet pectin solutions were found to besimilar at pH 4.5 and 7.0 (data not shown).

The beet pectin (0.1 wt %, pH 4.5) solutions initially had a relativelyhigh absorbance at 325 nm (0.84 cm⁻¹). In the absence of laccase, theabsorbance of the beet pectin solutions did not change significantlyover time (FIG. 2A). When laccase was added to the beet pectin solutionsthe absorbance decreased over time, which was attributed tocross-linking of the ferulic acid groups catalyzed by the enzyme. Theabsorbance decreased steeply during the first 250 to 2500 seconds(depending on laccase concentration) and then decreased more gradually.The initial slope in the relative absorbance with time (1/A(0)×dA(t)/dt)increased with increasing laccase concentration (FIG. 2B). There was nota large change in the reaction rate between 5 and 10 AU, so we used 5 AUfor the remainder of the experiments. At pH 7, the beet pectin solutionscontaining 5 AU laccase had a fairly similar initial absorbance at 325nm (0.79 cm⁻¹), but the decrease in relative absorbance with time wasmuch less than at pH 5: −0.16×10⁻³ s⁻¹. This affect can be attributed tothe fact that the optimum activity of laccase is around pH 2.5 to 5.5.

Additional information about the ability of laccase to crosslink beetpectin was obtained from gelation measurements. The influence of laccase(1 to 10 AU) on the gelation of 0.5 wt % citrus and beet pectinsolutions was ascertained by visual observation. No gelation wasobserved in the citrus pectin samples, even after a few days of storage,although a gradual increase in solution viscosity was observed duringstorage. On the other hand, gelation was observed in the beet pectinsamples within 2 hours of storage, with the gelation time decreasingwith increasing laccase concentration: 105, 90, 60 and 50 minutes for 1,2, 5 and 10 AU, respectively. These results support the UV-visiblemeasurements, indicating that there were sufficient ferulic acid groupspresent in the beet pectin to promote extensive cross-linking of thepolysaccharide molecules, but that there was much less ferulic acid inthe citrus pectin.

The purpose of these experiments was to establish the optimum beetpectin concentration required to form stable secondary emulsionscontaining β-Lg-pectin coated droplets. The electrical charge(ζ-potential) and mean particle diameter (d₃₂) of emulsions (1 wt % cornoil, 0.05 wt % β-Lg, 5 mM phosphate buffer, pH 4.5) containing differentbeet pectin concentrations (0 to 0.2 wt %) were measured 24 hours afterpreparation (FIGS. 3 and 4). In the absence of pectin, the electricalcharge on the emulsion droplets was slightly positive (+7 mV) at pH 4.5because the adsorbed β-Lg was below its isoelectric point (pI≈5). Theelectrical charge on the droplets changed from positive to negative asthe pectin concentration in the emulsions was increased (FIG. 3). Thenegative charge on the droplets reached a relatively constant value(≈−31 mV) when the pectin concentration exceeded about 0.04 wt %. Thesemeasurements indicated that negatively charged pectin molecules adsorbedto the surface of the β-Lg coated lipid droplets until the dropletsbecame saturated with polysaccharide.

The stability of the secondary emulsions was determined by static lightscattering, microscopy and creaming measurements (FIG. 4). The meanparticle diameter was relatively high (d₃₂>10 μm), large aggregates wereobserved in the photo-micrographs (d>10 μm), and rapid creaming (CI>70%)was observed in the emulsions containing 0 and 0.01 wt % beet pectin.These results can be attributed to the low droplet charge and/orextensive bridging flocculation of the cationic protein-coated dropletsby the anionic polysaccharide molecules. At pectin concentrations higherthan 0.02 wt % the emulsions appeared relatively stable to dropletaggregation (d₃₂<1 μm; no visible flocs; CI≈0%). These latter resultssuggest that there was sufficient pectin present to rapidly adsorb andfully cover the protein-coated lipid droplet surfaces thereby preventingbridging flocculation, but there was not so much non-adsorbed pectinremaining in the aqueous phase that it caused depletion flocculation.For these reasons, 0.04 wt % beet pectin was used in the remainder ofthe studies to form stable lipid droplets coated by aprotein-polysaccharide interfacial complex.

Studies were conducted to examine the impact of laccase catalyzedcross-linking of the adsorbed pectin molecules on the stability of thesecondary emulsions. A series of secondary emulsions were preparedcontaining either 0 or 5 AU of laccase (Section 2.4.2). The particlecharge and mean particle diameter of the emulsions were then measured atpH 7.0 (initial), pH 4.5 (laccase treatment) and pH 7.0 (final) (FIGS. 5and 6).

The protein-coated lipid droplets in the initial primary emulsion had arelatively high negative charge (ζ=−76 mV) at pH 7.0 because this pH wasabove the isoelectric point (pI) of the adsorbed β-Lg (pI≈5). Inaddition, the mean particle size was relatively small (z-averagediameter=0.21 μm), which indicated that the initial primary emulsion wasstable to droplet aggregation. When the primary emulsion was adjusted topH 4.5 the droplets became positively charged (ζ≈+2 mV) because this pHwas slightly below the pI of β-Lg (FIG. 5). In addition, extensivedroplet flocculation occurred at this pH (z-average diameter>6 μm),which can be attributed to the reduction of the electrostatic repulsionbetween the droplets (FIG. 6). When the primary emulsion was adjustedback to pH 7.0 the protein-coated lipid droplets again became negativelycharged (ζ=−55 mV), although the charge was appreciably less negativethan in the initial emulsion at pH 7.0 (ζ=−76 mV). This reduction in themagnitude of the charge can be attributed to the increase in ionicstrength associated with adding HCl and NaOH to adjust the pH from 7.0to 4.5 to 7.0. The droplets in the primary emulsion remained highlyaggregated (z-average diameter=3.2 μm) after the pH was adjusted from pH4.5 to pH 7.0, which indicates that the droplet aggregation thatoccurred at pH 4.5 was at least partially irreversible.

The droplets in the secondary emulsions were highly negatively chargedat pH 4.5 (in contrast to those in the primary emulsions), whichindicated that anionic pectin molecules adsorbed to positive patches onthe protein-coated lipid droplets (FIG. 5). The pectin molecules adsorbto the protein-coated droplet surfaces when the pH is decreased from 7to 4.5 due to electrostatic attraction between the anionic pectin andcationic patches on the protein surface. The addition of laccase causedno significant change (p<0.05) in the ζ-potential of the droplets in theemulsions. All of the secondary emulsions (0 and 5 AU laccase) werestable to extensive droplet aggregation at pH 4.5, with the z-averagediameter being ≈0.35 μm. These results suggest that the adsorption ofthe pectin molecules to the droplet surfaces stabilized them againstflocculation, which can be attributed to their ability to increase theelectrostatic and steric repulsion between the droplets, and reduce thevan der Waals attraction. The fact that the measured mean particlediameter was larger in the secondary emulsions at pH 4.5 than in theprimary emulsions at pH 7.0 can be attributed to the presence of theadsorbed polysaccharide layer around the protein-coated droplets or tosome droplet aggregation occurring during pH adjustment. When thesecondary emulsions were adjusted from pH 4.5 to pH 7.0 the particlesbecame highly negatively charged (FIG. 5) and they remained relativelysmall (FIG. 6), which suggested that they were stable to extensivedroplet aggregation. Unfortunately, the ζ-potential measurements at pH7.0 did not enable ascertaining whether the beet pectin moleculesremained attached to the droplet surfaces (as would be expected forcross-linked pectin), or whether they became detached (as would beexpected for non-cross-linked pectin). Such results indicate that beetpectin did adsorb to the protein-coated lipid droplets at pH 4.5 andstabilize them against aggregation.

It was hypothesized that beet pectin that was covalently cross-linked atthe droplet surfaces would remain attached when the pH was adjusted frompH 4.5 to pH 7.0, but pectin that was not covalently cross-linked wouldbecome detached because of the electrostatic repulsion between theanionic β-Lg and pectin molecules. If the pectin layer did remainattached to the droplet surfaces at pH 7, then the emulsions would beexpected to be more stable to salt because there would be a strongsteric repulsion between them and a weaker van der Waals attraction.Studies were conducted to examine the influence of salt (NaCl and CaCl₂)on the stability of primary and secondary emulsions (0 or 5 AU laccase)at pH 7.0.

The influence of NaCl concentration (0 to 500 mM) on the ζ-potential,mean particle diameter, and creaming stability of primary and secondaryemulsions at pH 7.0 was measured (FIG. 7A). In this series ofexperiments the primary emulsion was prepared and kept at pH 7.0 (toavoid aggregation at the isoelectric point), but the secondary emulsionswere prepared at pH 7.0, adjusted to pH 4.5, then brought back to pH7.0. The ξ-potential in the primary and secondary emulsions was negativeat all ionic strengths, but the magnitude of the ζ-potential decreasedas the NaCl concentration increased, which can be attributed toelectrostatic screening. The mean particle diameter (d₃₂) of the primaryemulsions increased appreciably when they contained ≧300 mM NaCl (FIG.7B). However, appreciable creaming instability was observed in theprimary emulsions at ≧100 mM NaCl (FIG. 7C). The difference in theminimum salt concentration required to promote emulsion instabilitybetween the particle size and creaming measurements can be attributed tothe fact that the emulsions had to be diluted appreciably for the lightscattering measurements, which may have disrupted any weak flocspresent.

Interestingly, the secondary emulsions containing no enzyme (0 AUlaccase) were less stable than the primary emulsions, exhibiting anappreciable increase in mean particle diameter and creaming instabilityat ≧50 mM NaCl. This effect can be attributed to the fact that there isfree (non-adsorbed) pectin present in these secondary emulsions at pH7.0, which will generate a depletion attraction between the droplets.Hence, the height of the repulsive energy barrier between the dropletswill be lower than in the absence of pectin, which would mean that lesssalt was required to induce aggregation. On the other hand, thesecondary emulsions containing enzyme (5 AU laccase) were much morestable to aggregation than the primary emulsions, showing no change inmean particle diameter and no creaming from 0 to 500 mM NaCl (FIG. 7).It is postulated that the adsorbed beet pectin was cross-linked bylaccase at pH 4.5, which prevented it from desorbing from the dropletsurfaces when the pH was adjusted to pH 7.0. Consequently, there was athick polymer layer around the lipid droplets, which increased thesteric repulsion and reduced the van der Waals attraction between thedroplets, thereby increasing their stability.

Similar experiments were carried out using CaCl₂ (0 to 200 mM) ratherthan NaCl (data not shown). It was found that the primary emulsion andthe secondary emulsion without laccase treatment were stable from 0 to 5mM CaCl₂, but were unstable to droplet aggregation and creaming at ≧8 mMCaCl₂. On the other hand, the secondary emulsion containing laccase wasstable from 0 to 8 mM CaCl₂, but unstable at ≧10 mM CaCl₂. The reasonthat a much lower concentration of CaCl₂ was required to promote dropletaggregation than NaCl is because the counter-ion was divalent (Ca²⁺),rather than monovalent (Na⁺). Multivalent counter-ions are much moreeffective at promoting droplet aggregation than monovalent ions for anumber of reasons: (i) they increase the ionic strength moreeffectively, thereby causing greater electrostatic screening; (ii) theybind to oppositely charged droplet surfaces, thereby reducing theircharge density; (iii) they can act as “salt-bridges” between oppositelycharged droplets. These results showed that covalently cross-linkingpectin with laccase provided some degree of protection against dropletaggregation in the presence of calcium ions, but was much less effectivethan for sodium ions. It is well known that calcium ions can crosslinkpectin molecules in aqueous solutions, and therefore it is notsurprising that calcium ions were able to promote droplet flocculationin the secondary emulsions.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compositions and/or methods of the presentinvention, including the interfacial assembly of oil in water emulsionsystems, as are available through the methodologies described herein. Incomparison with the prior art, the present compositions and methodsprovide results and data that are surprising, unexpected and contrarythereto. While the utility of this invention is illustrated through theuse of several emulsions, hydrophobic phases, components andcross-linking agents that can be used therewith, it will be understoodby those skilled in the art that comparable results are obtainable withvarious other emulsion systems, phases, components and/or cross-linkingagents, as are commensurate with the scope of this invention.

Materials

Powdered β-lactoglobulin (β-Lg) was kindly supplied by Davisco FoodsInternational (lot no. JE 003-3-922, Le Sueur, Minn.). The proteincontent was reported to be 98.3% (dry basis) by the supplier, with β-gmaking up 95.5% of the total protein. The moisture content of theprotein powder was reported to be 4.9%. Beet pectin was obtained fromHerbstreith & Fox K G (Elmsford, N.Y.) and citrus pectin was purchasedfrom Sigma-Aldrich Co. (St Louis, Mo.). As stated by the manufacturers,the degree of esterification (DE) of the citrus pectin and beet pectinwere respectively 60% and 50%. laccase enzyme (from Trametes versicolor)was purchased from Sigma-Aldrich Co. (lot no. 1210197 10306259,Steinheim, Germany). Laccase was reported to have 22.6 activity unitsper mg (AU) of enzyme. Corn oil was purchased from a local supermarketand used without further purification. Analytical grade hydrochloricacid, sodium hydroxide, sodium azide, and sodium phosphate were obtainedfrom Sigma-Aldrich (St. Louis, Mo.). Distilled and de-ionized water wasused for the preparation of all solutions.

Example 1 Solution Preparation

Stock buffer solutions were prepared by dispersing 5 mM disodiumhydrogen phosphate in distilled water and then adjusting the pH toeither 4.5 or 7.0 using 1 M HCl and/or 1 M NaOH. Citrus pectin and beetpectin solutions were prepared by dispersing 1 wt % powdered pectin intobuffer solutions at pH 7.0. An emulsifier solution was prepared bydispersing 0.5 wt % β-lactoglobulin powder into buffer solution at pH7.0. Enzyme solutions were prepared by dispersing 0.5 wt % laccasepowder into buffer solutions (pH 4.5 and 7.0). A sodium azide solution(an antimicrobial) was prepared by dispersing 0.04 wt % of its powderinto buffer solution (pH 7.0). Each solution was then stirred for atleast two hours to ensure complete dissolution of the materials.

Example 2 Characterisation of Laccase Activity

Establishment of Laccase Activity: UV-Visible Measurements.

Information about the ability of laccase to cross-link pectin moleculeswas obtained from UV-visible absorption measurements (UV-2101 PC,Shimadzu Corporation, Japan). Initially, absorption spectra of 0.1 wt %pectin (beet and citrus) dissolved in aqueous phosphate buffer solutionswere measured at pH 4.5 and pH 7.0, using buffer solutions containing nopectin as blanks. A maximum in the absorption spectrum was observed at awavelength of 325 nm for beet pectin, which was attributed to thepresence of the ferulic acid groups. Consequently, absorptionmeasurements at this wavelength (A_(325nm)) were used to establish theability of laccase to cross-link the pectin. Different amounts oflaccase (0 to 10 AU) were added to samples of beet pectin (0.1 wt %) atpH 4.5. The oxidation of ferulic acid was then followed by measuring theabsorbance at 325 nm at 25° C. for one and a half hours.

Example 3 Establishment of Laccase Activity

Pectin Gelation.

A qualitative indication of the ability of laccase to cross-link pectinmolecules was obtained by examining the enzymes influence on the gellingproperties of polysaccharide solutions. Solutions of citrus and beetpectin (0.5 wt %) were prepared in stock buffer solution at pH 4.5, thendifferent amounts of laccase were added (0 to 10 AU) and the resultingsolution was stirred for 30 minutes. The gelation state of the systemwas determined by visual inspection of the reaction mixture.Periodically, the vessel containing the reaction mixture was tilted andthe system was considered to have gelled when it did not deform underits own weight. The “gelation time” was then defined as the timerequired for a self-supporting gel to be formed.

The above experiments indicated that 5 AU of laccase were sufficient topromote cross-linking of the ferulic acid (see above): this amount wasused in subsequent experiments.

Example 4 Emulsion Preparation Optimum Conditions to FormProtein-Polysaccharide Coated Droplets

Preliminary experiments were carried out to determine the optimum beetpectin concentration required to create secondary emulsions. Primaryemulsions were prepared by homogenizing 10 wt % corn oil with 90 wt %aqueous emulsifier solution (0.5 wt % β-lactoglobulin in distilledwater, pH 7.0) in a high-speed blender (M133/1281-0, Biospec Products,Inc., ESGC, Switzerland) followed by five passes at 3000 psi through ahigh-pressure valve homogenizer (LAB 1000, APV-Gaulin, Wilmington,Mass.). The primary emulsion was then heated to 80° C. for 20 minutes tocovalently cross-link the adsorbed β-lactoglobulin molecules, therebyavoiding the possibility of any surface-active components in the beetpectin ingredient displacing the protein from the oil-water interface.The droplets did not aggregate during this process because at low ionicstrengths there is a strong electrostatic repulsion between them.

Secondary emulsions were formed by mixing primary emulsions with aqueousbeet pectin solutions at pH 7.0 for 10 minutes using a magnetic stirrerto produce a series of emulsions with different pectin concentrations: 1wt % corn oil, 0.05 wt % β-Lg, and 0 to 0.2 wt % beet pectin. The pH wasthen adjusted to pH 4.5 with 1 M HCl and the emulsions were stirred for20 minutes using a magnetic stirrer. The resulting emulsions were thenstored at room temperature for 24 h before being analyzed. Theζ-potential, particle size distribution and creaming stability of theemulsions were then measured (see below). The optimum beet pectinconcentration required to form non-aggregated lipid droplets coated bypectin was determined to be 0.04 wt % (see below), so that this amountwas used to prepare the secondary emulsions in all subsequentexperiments.

Example 5 Influence of Laccase on the Stability of Secondary Emulsions

A purpose of these experiments was to study the impact of laccasecatalyzed cross-linking of the adsorbed pectin molecules on thestability and properties of secondary emulsions. The presentmethodologies were used to prepare secondary emulsions containing 1 wt %corn oil, 0.05 wt % cross-linked β-Lg and either 0 or 0.04 wt % pectin(5 mM phosphate buffer, pH 7.0). These emulsions were then adjusted topH 4.5 using 1 M HCl, and then either 0 or 5 AU laccase was added. Theemulsions were then stored for 24 hours at ambient temperature prior toanalysis. A series of emulsions were prepared using this approach tostudy the influence of pectin and laccase on their properties: (i)“control”—0 wt % pectin, 0 AU laccase; (ii) “beet (0 AU)”—0.04 wt % beetpectin, 0 AU laccase; and (iii) “beet (5 AU)”—0.04 wt % beet pectin, 5AU laccase.

Example 6 Salt Stability of Emulsions

The influence of salt addition on the stability of the primary andsecondary emulsions was examined at pH 7. Primary emulsions (0 wt %pectin) and secondary emulsions (0.04 wt % beet pectin) were prepared atpH 7.0. The emulsions were then adjusted to pH 4.5 using 1M HCl andeither 0 or 5 AU of laccase were added. After 24 hours storage atambient temperature, the emulsions were adjusted to pH 7.0 with 1M NaOH.NaCl (0 to 500 mM) or CaCl₂ (0 to 200 mM) was then added to theemulsions and they were stored for a further 24 hours at roomtemperature prior to analysis.

Example 7 Particle Size Measurements

The particle size distribution of the emulsions was measured using bothstatic light scattering (Mastersizer MSS, Malvern Instruments,Worcestershire, UK) and dynamic light scattering (Zetasizer Nano-ZS,Malvern Instruments, Worcs., UK). For both techniques, the emulsionswere diluted prior to analysis using an appropriate phosphate buffersolution to avoid multiple scattering effects. The mean particle sizewas reported as the surface-weighted mean diameter (d₃₂=Σn_(i)d_(i)³/Σn_(i)d_(i) ²) for the static light scattering measurements, and theintensity-weighted mean diameter (z-average) for the dynamic lightscattering measurements. The static light scattering measurements weremainly used for highly flocculated emulsions containing large particles(d>10 μm), whereas the dynamic light scattering was mainly used fornon-flocculated emulsions containing small particles (d<1 μm).

Example 8 ξ-Potential Measurements

Emulsions were diluted to a droplet concentration of approximately 0.005wt % using an appropriate buffer solution to avoid multiple scatteringeffects. Diluted emulsions were injected directly into the measurementchamber of a particle electrophoresis instrument (ZEM5002, Zetamaster,Malvern Instruments, Worcestershire, UK) that measured the direction andvelocity of particle movement in the applied electric field. Anindividual ξ-potential measurement was determined from the average offive readings taken on the same sample.

Example 9 Optical Microscopy

Emulsions were gently agitated in a glass test tube before analysis toensure that they were homogenous. A drop of emulsion was then placed ona microscope slide and covered with a cover slip. The microstructure ofthe emulsion was then observed using conventional optical microscopy(Nikon microscope Eclipse E400, Nikon Corporation, Japan). The imageswere acquired using a CCD camera (CCD-300T-RC, DAGE-MTI, Michigan City,Ind.) connected to Digital Image Processing Software (Micro VideoInstruments Inc., Avon, Mass.) installed on a computer.

Example 10 Creaming Stability Measurement

Ten grams of emulsion were transferred into a test tube (internaldiameter 15 mm, height 125 mm), tightly sealed with a plastic cap, andthen stored for 1 day at room temperature. After storage, emulsionsseparated into an optically opaque ‘cream’ layer at the top and atransparent (or turbid) ‘serum’ layer at the bottom. As defined, theserum layer is the sum of the turbid and transparent layers. The totalheight of the emulsions (H_(E)) and the height of the serum layer (Hs)were measured. The extent of creaming was characterized by creamingindex (%)=100×(H_(S)/H_(E)). The creaming index provided indirectinformation about the extent of droplet aggregation in an emulsion: thefaster the creaming, the larger the particle size.

Example 11 Statistical Analysis

Experiments were performed at least twice using freshly preparedsamples. Averages and standard deviations were calculated from thesemeasurements.

Demonstrating various aspects of this invention, the preceding examplesshow that laccase can be used to covalently cross-link beet pectinmolecules adsorbed to the surfaces of protein-coated lipid droplets atpH 4.5. Results show that the beet pectin layer remains attached to thedroplet surfaces when the pH is raised from 4.5 to 7.0 (e.g., as thecomposition is partially neutralized or neutralized), even though itwould normally be expected to become detached because of theelectrostatic repulsion between the anionic pectin and anionicprotein-coated droplets at pH 7. Emulsions containing lipid dropletscoated by β-lactoglobulin and cross-linked beet pectin had much betterstability to salt (NaCl) than those coated by β-lactoglobulin alone,which can be attributed to the ability of the adsorbed pectin layer toincrease the repulsive interactions and decrease the attractiveinteractions between the droplets. These results show that emulsionswith improved functional performance can be prepared via a bio-mimeticapproach, utilizing enzymes to cross-link adsorbed biopolymers.

While various principles relating to this invention have been describedin conjunction with certain embodiments, it should be understood clearlythat these descriptions are presented only by way of example and are notintended to limit, in any way, the scope of this invention. Forinstance, as would be understood by those skilled in the art made awareof this invention, the various methods and compositions of the presentinvention can also be directed to use of biopolymers such as agar,carrageenan, chitosan, and gelatin which can be cross-linked withgenipin. Various proteins and carbohydrates can also be cross-linked byheating in conjunction with the Maillard reaction, after emulsionformation and corresponding biopolymer contact. Likewise, certainproteins, as would be understood by those skilled in the art, can becross-linked with various other enzymes, including but not limited to atransglutaminase, by heating, or by introduction of certain othercross-linking compounds or reagents, such as but not limited togluteraldehyde. Various other features and advantages of the presentinvention will become apparent from the claims hereinafter, as would beunderstood by those skilled in the art.

1-20. (canceled)
 21. A biomimetic method of using a cross-linkablebiopolymer to prepare a stabilized emulsion composition, said methodcomprising: encompassing each droplet of a hydrophobic component with anemulsifier component; the hydrophobic component being formed intodroplets and being in an aqueous medium; and adsorbing a biopolymercomponent on said emulsifier component that encompasses each droplet ofthe hydrophobic component; the biopolymer component comprising anintra-component covalent cross-linkage, said cross-linkage the reactionproduct of two cross-linkable moieties and a cross-linking reagent; atleast a portion of said emulsifier component comprising a net charge andat least a portion of said biopolymer component comprising another netcharge opposite the net charge of said emulsifier component, saidemulsifier component and said biopolymer component beingelectrostatically interactive.
 22. The method of claim 21 wherein saidcross-linking agent is selected from heat, pH, a redox reagent and anenzyme.
 23. The method of claim 21 wherein said biopolymer component isselected from pectins comprising ferulic acid moieties, proteinscomprising phenolic acid moieties and combinations thereof.
 24. Themethod of claim 23 wherein said biopolymer component is a beet pectin.25. The method of claim 23 wherein said biopolymer moieties arecross-linked with an enzyme.
 26. The method of claim 25 wherein saidbiopolymer is a beet pectin.
 27. (canceled)
 28. The method of claim 27wherein said emulsifier component is a protein component comprising anet positive charge, said composition at a pH below the isoelectricpoint of said protein.
 29. The method of claim 28 wherein saidbiopolymer component is a polysaccharide comprising a net negativecharge.
 30. The method of claim 29 wherein said emulsifier component isa whey protein comprising phenolic acid moieties, and said biopolymercomponent is a pectin comprising ferulic acid moieties; and saidcross-linking agent is selected from a laccase enzyme, a peroxidaseenzyme and combinations of said enzymes, to provide covalentintra-component cross-linkages, covalent inter-component cross-linkagesand combinations thereof.
 31. The method of claim 27 comprisingadjusting pH of said composition to at least partially neutralize thenet charge of at least one said component.
 32. The method of claim 27comprising iterative contact of said emulsifier and biopolymercomponents.
 33. The method of claim 21 wherein said hydrophobiccomponent is selected from fatty acids, poly-unsaturated fatty acids,triglycerides and functional derivatives thereof, carotenoids, terpenes,anti-oxidants, colorants, flavor oils, food oils, fat-soluble vitamins,nutrients, nutraceuticals and combinations thereof.
 34. The method ofclaim 21 comprising incorporation into one of a food product, abeverage, a nutraceutical product, a cosmetic product and apharmaceutical product.
 35. A method for emulsion preparation, saidmethod comprising: providing a lipid-protein emulsification productcomprising a lipid component and a protein component; the lipidcomponent being formed into number of droplets; the protein componentencompassing each droplet of the lipid component of the lipid component;and adsorbing a polysaccharide biopolymer component on the proteincomponent that encompasses each droplet of the lipid component; thepolysaccharide biopolymer component comprising an intra-componentcovalent cross-linkage, said cross-linkage the reaction product of twocross-linkable moieties and a cross-linking reagent and.
 36. The methodof claim 35 wherein said polysaccharide biopolymer component is selectedfrom pectins comprising ferulic acid moieties and combinations thereof,said polysaccharide biopolymer component cross-linked with an enzyme.37. The method of claim 36 wherein said polysaccharide biopolymercomponent is a beet pectin.
 38. The method of claim 35 wherein saidemulsifier component is selected from dairy proteins and combinationsthereof.
 39. The method of claim 38 wherein said emulsifier component isa whey protein comprising phenolic acid moieties, and saidpolysaccharide biopolymer component is a pectin comprising ferulic acidmoieties; and said cross-linking agent is selected from a laccaseenzyme, a peroxidase enzyme and combinations of said enzymes, to providecovalent intra-component cross-linkages, covalent inter-componentcross-linkages and combinations thereof.
 40. The method of claim 35wherein said lipid component is selected from fatty acids,poly-unsaturated fatty acids, triglycerides and functional derivativesthereof, carotenoids, terpenes, anti-oxidants, colorants, flavor oils,food oils, fat-soluble vitamins, nutrients, nutraceuticals andcombinations thereof, said emulsification product incorporated into oneof a food product, a beverage, a nutraceutical product, a cosmeticproduct and a pharmaceutical product.